Uropathogenic E. coli (UPEC) are the leading cause of all types of urinary tract infections (UTIs): cystitis and pyelonephritis, community- acquired and nosocomial, complicated and uncomplicated, symptomatic and asymptomatic. Despite many of study, a clear understanding of the pathogenesis of UPEC infections has not emerged. With the exception of type-1 and P fimbriae, heretofore suspected virulence factors have not been confirmed to be essential for UT in animal models. Meanwhile, the discovery the pathogenicity islands in UPEC strains has awakened our appreciation that vast stretches of the genomes of these organisms have yet to be scrutinized. We propose a trifaceted approach to accomplish signature-tagged mutagenesis to identify genetic loci required for colonization in our murine model of ascending UTI. Our success thus far in isolating 12 such mutants portends well for the achievement of this aim. Our second specific aim is to identify fragments of E. coli mutants portends well for the achievement of this aim. Our second specific aim is to identify fragments of E. coli CFT073 genome that are not present in E. coli K-12. Our second specific aim is to identify fragments of E. coli CFT073 genome that are not present in E. coli K-12. We have used a PCR-based genome subtraction approach to clone fragments enriched for such sequences and, using this approach have identified a gene encoding the usher from a previously unknown fimbria. Our third specific aim is to use a green fluorescent protein promoter trap and a fluorescence activated cell sorter to identify promoters from E. coli CFT073 that are expressed specifically in the murine urinary tract. Finally, we will prioritize the loci identified in the first three specific aims and select the most promising for further study in vivo and in vitro to determine the mechanisms by which the products these genes contribute to the pathogenesis of UTI. We believe that this comprehensive analysis of the virulence determinants of UPEC will greatly advance our understanding of the pathogenesis of UTI.